Antibody adsorbent with immobilized Fc receptor

Abstract

To provide a Fc receptor-immobilized antibody adsorbent that has improved antibody adsorption capacity than before and can be used for the purpose of antibody fractionation.SOLUTION: By an antibody adsorbent in which a Fc receptor is immobilized on an insoluble carrier via a peptide linker having a length of 2 nm or longer, the problem is solved.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an antibody adsorbent having an Fc receptor immobilized thereon, and more particularly to such an adsorbent that has an improved antibody adsorption capacity compared to conventional adsorbents. [Background technology]

[0002] Antibody drugs are medicines that utilize antibodies (immunoglobulins), molecules that are responsible for the body's immune functions. Antibody drugs bind to target molecules with high specificity and affinity due to the diversity of the variable regions of antibodies. As a result, antibody drugs have few side effects, and in recent years, the range of diseases for which they are suitable has been expanding, leading to a rapid expansion of the market.

[0003] The sugar chain structure of antibody drugs is known to be significantly related to their efficacy and stability. Therefore, controlling the sugar chain structure is extremely important when manufacturing antibody drugs. Among antibody-binding proteins, the Fc receptor FcγRIIIa is known to recognize the sugar chain structure of antibodies (immunoglobulins). Antibodies can be separated based on their sugar chain structure by using an adsorbent in which FcγRIIIa is immobilized on an insoluble carrier as an affinity ligand (Patent Document 1). Therefore, this adsorbent is useful for process analysis during the manufacturing of antibody drugs. Furthermore, by creating a mutant of FcγRIIIa in which an amino acid residue at a specific position in the extracellular domain (specifically, the region from glycine at position 17 to glutamine at position 192 in the amino acid sequence set forth in SEQ ID NO: 1) is substituted with another specific amino acid residue, the thermal stability, acid stability, and alkaline stability required for an affinity ligand are improved (Patent Documents 1 to 3).

[0004] However, when attempts were made to apply an antibody adsorbent comprising an insoluble carrier and FcγRIIIa or the variant immobilized on the carrier to the purpose of separating antibodies based on their sugar chain structures, the amount of antibody adsorbed was insufficient, making it difficult to apply the adsorbent to the purpose of separating antibodies in the industrial production of antibody pharmaceuticals.

[0005] Patent Document 4 discloses that antibody adsorption capacity is improved by immobilizing an antibody-binding protein (Protein A derived from Staphylococcus aureus) on an insoluble carrier via a peptide linker containing polyproline and having a length of 0.9 to 91 nm. However, there has been insufficient research into the use of peptide linkers to improve adsorption capacity in antibody adsorbents on which Fc receptors are immobilized. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2015-086216 [Patent Document 2] Japanese Patent Application Laid-Open No. 2016-169197 [Patent Document 3] Japanese Patent Application Publication No. 2017-118871 [Patent Document 4] WO2017 / 018437 issue Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide an antibody adsorbent having an immobilized Fc receptor, which has an improved antibody adsorption capacity compared to conventional adsorbents and can also be used for antibody fractionation. [Means for solving the problem]

[0008] As a result of extensive research to solve the above problems, the length of the peptide linker used to immobilize the Fc receptor on the insoluble support was optimized, resulting in an antibody adsorbent with improved antibody adsorption capacity compared to conventional adsorbents.

[0009] That is, the present invention includes the following aspects [1] to [5].

[0010] [1] An antibody adsorbent comprising an Fc receptor, an insoluble carrier, and a peptide linker for immobilizing the receptor on the carrier, wherein the length of the peptide linker is 2 nm or more.

[0011] [2] The adsorbent according to [1], wherein the peptide linker is a polypeptide shown in either (1) or (2) below: (1) A polypeptide containing at least the amino acid residues from proline at position 24 to histidine at position 105 in the amino acid sequence set forth in SEQ ID NO: 1; (2) A polypeptide having an amino acid sequence comprising at least the amino acid residues from the 24th proline to the 105th histidine in the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the 24th to 105th amino acid residues further include substitution, deletion, insertion, or addition of one or more amino acid residues at one or more positions.

[0012] [3] The adsorbent according to [2], wherein the peptide linker is a polypeptide containing at least the amino acid residues from the 6th proline to the 87th histidine in the amino acid sequence set forth in SEQ ID NO: 17.

[0013] [4] The adsorbent according to any one of [1] to [3], wherein the Fc receptor is a polypeptide shown in either (i) or (ii) below: (i) a polypeptide containing at least the amino acid residues from glycine at position 17 to glutamine at position 192 in the amino acid sequence set forth in SEQ ID NO: 1; (ii) A polypeptide having an amino acid sequence comprising at least the amino acid residues from glycine at position 17 to glutamine at position 192 of the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the amino acid sequence further comprises substitution, deletion, insertion or addition of one or more amino acid residues at one or more positions within the amino acid residues at positions 17 to 192, and having binding activity to an antibody.

[0014] [5] A method for purifying an antibody, comprising the steps of: adding a solution containing an antibody to a column packed with the adsorbent according to any one of [1] to [4] to adsorb the antibody onto the adsorbent; and eluting the antibody adsorbed onto the adsorbent using an elution solution.

[0015] The present invention will be described in detail below.

[0016] In the present invention, the Fc receptor immobilized on an insoluble carrier is a protein that has binding affinity to the Fc region of an antibody (immunoglobulin). Specific examples of human Fc receptors include human FcγRI, human FcγRIIa, human FcγRIIb, human FcγRIIIa, and human FcRn. In particular, human FcγRIIIa is a human Fc receptor that can recognize the sugar chain structure of antibodies. An antibody separating agent in which human FcγRIIIa is immobilized on an insoluble carrier can separate antibodies based on their sugar chain structure (Japanese Patent Laid-Open Publication No. 2015-086216). Therefore, this is a preferred embodiment of the Fc receptor to be immobilized on an insoluble carrier in the present invention.

[0017] A preferred embodiment of human FcγRIIIa is (i) a polypeptide comprising at least the extracellular region of native human FcγRIIIa (amino acid residues from glycine at position 17 to glutamine at position 192 in the amino acid sequence set forth in SEQ ID NO: 1); and (ii) a polypeptide comprising at least the extracellular region of native human FcγRIIIa, and further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions among the amino acid residues constituting the extracellular region, and having antibody-binding activity; Furthermore, a more preferred embodiment of the above (ii) is as follows: Fc-binding proteins disclosed in JP 2015-086216 A; Fc-binding proteins disclosed in JP 2016-169197 A; Fc-binding proteins disclosed in JP 2017-118871 A; Fc-binding proteins disclosed in JP 2018-197224 A; Fc-binding proteins disclosed in WO2019 / 083048; Examples include:

[0018] In the present invention, the insoluble support on which an Fc receptor is immobilized may be any material that has an activated functional group capable of immobilizing a peptide linker (described below) and is insoluble in the solution or solvent used for adsorption / elution of the antibody, and may be a support derived from an inorganic substance such as zirconia, zeolite, silica, or coated silica; a support derived from a natural organic polymer such as cellulose, agarose, or dextran; or a support derived from a synthetic organic polymer such as polyacrylic acid, polystyrene, polyacrylamide, polymethacrylamide, polymethacrylate, or vinyl polymer. Furthermore, the activated functional group may be bound to a known chromatography support such as Shodex (Showa Denko K.K.), Sepharose (Cytiva Corporation), Amberlite (DuPont), Cellufine (JNC), POROS (Thermo Fisher Scientific), or Toyopearl (Tosoh Corporation).

[0019] The adsorbent of the present invention is a compound for immobilizing the Fc receptor on the insoluble carrier. The peptide linker is characterized in that the length of the peptide linker is 2 nm or more. The length of the peptide linker is preferably 2 nm or more and 10 nm or less, and more preferably 3 nm or more and 8 nm or less. The sequence of the polypeptide constituting the peptide linker is not particularly limited as long as it satisfies the above-mentioned length condition. Examples include a sequence in which Pro (proline)-Ala (alanine) is repeated 8 or more times, a sequence in which Gly (glycine)-Gly-Ser (serine) is repeated 5 or more times, and a sequence in which Glu (glutamic acid)-Ala (alanine)-Ala-Ala-Lys (lysine) (SEQ ID NO: 2) is repeated 3 or more times.

[0020] A partial sequence of an Fc receptor may be used as the peptide linker. A preferred embodiment of a partial sequence of an Fc receptor as the peptide linker is a polypeptide comprising the D1 domain of human FcγRIIIa. Specifically, (1) A polypeptide containing at least the amino acid residues from proline at position 24 to histidine at position 105 of the amino acid sequence set forth in SEQ ID NO: 1; or (2) A polypeptide having an amino acid sequence comprising at least the amino acid residues from the 24th proline to the 105th histidine in the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the 24th to 105th amino acid residues further include substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions. A preferred embodiment of (2) above is a polypeptide containing at least the amino acid residues from proline at position 6 to histidine at position 87 in the amino acid sequence set forth in SEQ ID NO: 17.

[0021] An oligopeptide for immobilization on an insoluble support may be further added to the C-terminus of the peptide linker. Examples of the oligopeptide include polyhistidine, polylysine, polyarginine, polyglutamic acid, polyaspartic acid, and a cysteine ​​tag consisting of the amino acid sequence set forth in SEQ ID NO: 3.

[0022] The adsorbent of the present invention can be produced by preparing a polypeptide in which the aforementioned peptide linker has been added to the C-terminus of an Fc receptor, and then immobilizing the polypeptide on an insoluble support. When adding a peptide linker to an Fc receptor, a polynucleotide encoding the peptide linker can be prepared and then genetically engineered using methods well known to those skilled in the art. Alternatively, a chemically synthesized peptide linker can be chemically bonded to the C-terminus of the Fc receptor of the present invention. Furthermore, a signal peptide may be added to the N-terminus of the Fc receptor to promote efficient expression in a host. Examples of the signal peptide when the host is E. coli include signal peptides that cause protein secretion into the periplasm, such as PelB (an oligopeptide consisting of amino acid residues 1 to 22 of UniProt No. P0C1C1), DsbA, MalE (an oligopeptide consisting of amino acid residues 1 to 26 of UniProt No. P0AEX9), and TorT (JP 2011-097898 A). Alternatively, the DsbC signal peptide (the region consisting of amino acids 1 to 21 of UniProt No. P0AEG7) or an oligopeptide derived from the signal peptide by deleting one or several amino acid residues other than the N-terminus (methionine at position 1) and the C-terminus (alanine at position 20 and aspartic acid at position 21) may be added to the N-terminus of the ligand.

[0023] To immobilize an Fc receptor having a peptide linker attached thereto on an insoluble support, an active group such as an N-hydroxysuccinimide (NHS)-activated ester group, an epoxy group, a carboxy group, a maleimide group, a haloacetyl group, a tresyl group, a formyl group, or a haloacetamide group may be attached to the insoluble support, and the peptide linker may be covalently bonded to the insoluble support via the active group. Supports having active groups attached thereto may be prepared, for example, by introducing the active groups onto the support surface under appropriate reaction conditions.

[0024] On the other hand, an example of a method for introducing an active group onto the surface of a carrier is a method in which one of a compound having two or more active sites is reacted with a hydroxy group, epoxy group, carboxy group, amino group, etc. present on the surface of the carrier. Examples of such compounds that introduce an epoxy group into a hydroxy group or amino group on the surface of the carrier include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether. After introducing epoxy groups onto the carrier surface using the above compound, compounds for introducing carboxy groups onto the carrier surface include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3-aminopropionic acid, and 4-aminobutyric acid. Compounds for introducing maleimide groups into hydroxy groups, epoxy groups, carboxy groups, and amino groups present on the carrier surface include N-(ε-maleimidocaproic acid) hydrazide, N-(ε-maleimidopropionic acid) hydrazide, 4-[4-N-maleimidophenyl]acetic acid hydrazide, 2-aminomaleimide, 3-aminomaleimide, 4-aminomaleimide, 6-aminomaleimide, 1-(4-aminophenyl)maleimide, and 1-(4-aminophenyl)maleimide. Examples include -(3-aminophenyl)maleimide, 4-(maleimido)phenyl isocyanate, 2-maleimidoacetic acid, 3-maleimidopropionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, N-(α-maleimidoacetoxy)succinimide ester, (m-maleimidobenzoyl)N-hydroxysuccinimide ester, succinimidyl-4-(maleimidomethyl)cyclohexane-1-carbonyl-(6-aminohexanoic acid), succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylic acid, (p-maleimidobenzoyl)N-hydroxysuccinimide ester, and (m-maleimidobenzoyl)N-hydroxysuccinimide ester.

[0025] Examples of compounds that introduce haloacetyl groups into hydroxy groups or amino groups present on the support surface include chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic acid bromide, chloroacetic acid anhydride, bromoacetic acid anhydride, iodoacetic acid anhydride, 2-(iodoacetamido)acetic acid-N-hydroxysuccinimide ester, 3-(bromoacetamido)propionic acid-N-hydroxysuccinimide ester, and 4-(iodoacetyl)aminobenzoic acid-N-hydroxysuccinimide ester. Another example of a method involves reacting hydroxy groups or amino groups present on the support surface with an ω-alkenylalkane glycidyl ether, followed by halogenating and activating the ω-alkenyl moiety with a halogenating agent. Examples of the ω-alkenylalkane glycidyl ether include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether, and examples of the halogenating agent include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide.

[0026] Another example of a method for introducing active groups onto the support surface is to use a condensing agent and an additive to introduce active groups into carboxyl groups present on the support surface. Examples of condensing agents include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole. Examples of additives include N-hydroxysuccinimide (NHS), 4-nitrophenol, and 1-hydroxybenzotriazole.

[0027] Examples of buffers that can be used when immobilizing Fc receptors on insoluble carriers include acetate buffer, phosphate buffer, MES (2-morpholinoethanesulfonic acid) buffer, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, Tris buffer, and borate buffer. The reaction temperature during immobilization can be appropriately set within the range of 5°C to 50°C, taking into consideration the reactivity of the active group and the stability of the Fc receptor, and is preferably in the range of 10°C to 35°C.

[0028] To separate antibodies using the antibody adsorbent of the present invention obtained by immobilizing Fc receptors on an insoluble carrier using the method described above, an equilibration solution is added to a column packed with the antibody adsorbent using a liquid delivery means such as a pump to equilibrate the column, an antibody-containing solution is added using the liquid delivery means to specifically adsorb the antibody to the antibody adsorbent, and then an appropriate eluent is added using the liquid delivery means to elute the adsorbed antibody. Note that in the present invention, antibody separation is not limited to separation of antibodies from a solution containing impurities (removal of impurities) but also includes separation of antibodies based on their structure, properties, activity, etc.

[0029] Examples of the equilibration solution include phosphate buffer, acetate buffer, MES buffer, and citrate buffer, and the buffer may further contain 10 mM to 100 mM (preferably 40 mM to 60 mM) of an inorganic salt such as sodium chloride. The pH of the equilibration solution may be optimally determined from a range of 4.0 to 7.0, taking into consideration the buffer components, column shape, and the packing pressure of the adsorbent into the column.

[0030] Antibodies adsorbed to antibody adsorbents can be eluted by weakening the interaction between the antibody and Fc receptor. Specific examples include lowering the pH with a buffer, adding a counter peptide, increasing the temperature, and changing the salt concentration. Specific examples of elution solutions for eluting antibodies adsorbed to antibody adsorbents include buffer solutions that are more acidic than the solution used to adsorb the antibody to the antibody adsorbent. Examples of such buffer solutions include citrate buffer, glycine hydrochloride buffer, and acetate buffer, which have acidic buffering capacity. The pH of the elution solution may be set within a range that does not impair the functions of the antibody (e.g., antigen binding). Examples include pH 2.5 to 6.0, pH 3.0 to 5.0, and pH 3.0 to 4.0.

[0031] When eluting antibodies by changing salt concentration, they may be eluted in a single step with a buffer solution (eluent) containing a high salt concentration, or by increasing the salt concentration stepwise (step gradient), or by increasing the salt concentration in a linear gradient (linear gradient), with linear gradient elution being preferred. For example, when using sodium chloride as the water-soluble salt, elution can be performed using a linear gradient from 0M to 1M sodium chloride. When eluting antibodies by changing pH, they may be eluted in a single step with an acidic buffer solution (eluent) with a lower pH than the equilibration buffer, or by decreasing the buffer pH stepwise (step gradient), or by decreasing the buffer pH in a linear gradient (linear gradient), with linear gradient elution being preferred. For example, elution can be performed using a linear gradient from a neutral to weakly acidic buffer solution to which the antibody adsorbs to an acidic buffer solution to which the antibody elutes.

[0032] The antibody can be obtained by collecting the antibody-containing fractions eluted by the above-mentioned method. Collection may be performed by a conventional method. Specific examples include a method of changing the collection container at regular intervals or at regular volume intervals, a method of changing the collection container according to the shape of the chromatogram of the eluate, and a method of collecting fractions using an automatic fraction collector. [Effects of the Invention]

[0033] The present invention provides an antibody adsorbent comprising an Fc receptor, an insoluble carrier, and a peptide linker for immobilizing the receptor on the carrier, wherein the peptide linker has a length of 2 nm or more. The adsorbent of the present invention exhibits a higher antibody adsorption capacity than conventional adsorbents with immobilized Fc receptors that do not contain the peptide linker. Therefore, the present invention is useful for antibody fractionation in the industrial production of antibody pharmaceuticals.

[0034] In particular, when human FcγRIIIa is used as the Fc receptor, antibody separation agents obtained by immobilizing these proteins on an insoluble carrier can separate antibodies based on their glycan structure (Patent Publication No. 2015-086216) or on the strength of their antibody-dependent cellular cytotoxicity (Patent Publication No. 2016-023152), making it possible to selectively and mass-produce antibodies with specific glycan structures or antibodies with high (or low) antibody-dependent cellular cytotoxicity. [Example]

[0035] The present invention will be explained in more detail below using comparative examples and examples, but the present invention is not limited to these examples.

[0036] Example 1 Preparation of insoluble carrier (1) Introduction of epoxy groups into gels containing hydroxyl groups (1-1) A suction-dried gel was prepared by filtering a slurry of polymethacrylate gel (Toyopearl, manufactured by Tosoh Corporation) having hydroxyl groups on its surface through a glass filter and then drying it by suction. (1-2) 5 mL of water, 0.5 mL of 0.2 M aqueous sodium hydroxide solution, and 5 mmol of an epoxidizing reagent (ethylene glycol diglycidyl ether) were added to 1.0 g of the suction-dried gel, and the mixture was stirred and shaken for 16 hours in a stirring shaker set at 50°C. (1-3) After the reaction was completed, the reaction solution was filtered through a glass filter and washed four times with water and four times with 1,4-dioxane to produce a gel with an epoxy group introduced therein (hereafter referred to as Epoxy Toyopearl).

[0037] (2) Introduction of haloacetyl groups into Epoxy Toyopearl (2-1) 1.0 g of Epoxy Toyopearl (suction-dried gel) obtained in (1) was added with 3.5 mmol of an amination reagent (tris(2-aminoethyl)amine) and 1 mL of water, and the mixture was stirred and shaken for 4 hours in a stirring shaker set at 45°C. (2-2) After the reaction of (2-1) was completed, the reaction solution was filtered through a glass filter and washed four times with water and four times with 1,4-dioxane to prepare a gel with introduced amino groups (hereafter referred to as Amino Toyopearl). (2-3) To 1.0 g of Amino Toyopearl (suction-dried gel), 7.5 mL of 1,4-dioxane, 2.5 mL of water, 0.5 mmol of EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide), and 0.5 mmol of a haloacetylating reagent (iodoacetic acid) were added, and the mixture was stirred and shaken for 2 hours in a stirring shaker set at 25°C. After the reaction of (2-4)(2-3) was completed, the reaction solution was filtered through a glass filter and washed four times with 1,4-dioxane and four times with water to prepare a gel containing haloacetyl groups (hereafter referred to as haloacetyl Toyopearl).

[0038] Comparative Example 1 Preparation of antibody adsorbent (part 1) The polypeptide FcR36i_Cys (JP 2020-092689 A) (SEQ ID NO: 4) containing the Fc receptor FcR36i was immobilized on an insoluble carrier to prepare an antibody adsorbent by the method described below. Note that the sequence from the 1st methionine to the 22nd alanine in FcR36i_Cys (SEQ ID NO: 4) is an improved PelB signal peptide (UniProt An oligopeptide consisting of the amino acid residues 1 to 22 of No. P0C1C1, with the exception that an amino acid substitution of proline at position 6 with serine has occurred), and the amino acid sequence from glycine at position 24 to glutamine at position 199 is the amino acid sequence of the Fc receptor FcR36i (the amino acid residues from glycine at position 17 to glutamine at position 192 of the amino acid sequence set forth in SEQ ID NO: 1, with the exception that the amino acid residues at positions 17 to 192 are Glu21Gly (this notation indicates that glutamic acid at position 21 has been substituted with glycine, the same applies below), Leu23Met, Val27Glu, Phe29Ile, Gln33Pro, Tyr35Asn, Lys40Gln, Gln48Arg, Tyr51Hi a polypeptide in which the following amino acid substitutions have occurred: Glu54Asp, Asn56Asp, Ser65Arg, Ser68Pro, Tyr74Phe, Phe75Ile, Ala78Ser, Thr80Ser, Asn92Ser, Val117Glu, Lys119Val, Glu121Gly, Asp122Glu, Lys132Arg, Thr140Met, Tyr141Phe, Gly147Val, Tyr158Val, Lys165Glu, Phe171Ser, Val176Ile, Ser178Arg, Asn180Lys, Glu184Gly, Thr185Ala, Asn187Asp, and Ile190Val; and the sequence from the 201st cysteine ​​to the 207th glycine is a cysteine ​​tag sequence (SEQ ID NO: 3).

[0039] (1) Preparation of FcR36i_Cys (1-1) Recombinant Escherichia coli containing the FcR36i_Cys expression vector pTrc-FcR36i_Cys, prepared by the method described in JP 2020-092689 A, was inoculated into 100 mL of 2xYT liquid medium (tryptone 1.6% (w / v), yeast extract 1% (w / v), sodium chloride 0.5% (w / v)) containing 50 μg / mL of kanamycin, and pre-cultured (37°C, 16 hours). (1-2) 10 mL of the culture broth after pre-culture was inoculated into 1 L of 2xYT liquid medium containing 50 μg / mL kanamycin and cultured at 37°C for 2 hours. After that, 1 mL of 0.01 M IPTG (Isopropyl-β-D-thiogalactopyranoside) was added and the mixture was further cultured overnight at 25°C with shaking. (1-3) After the cultivation was completed, the cells were collected by centrifugation and suspended in 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl. The cells were disrupted using an ultrasonic generator (Insonator 201M (trade name), manufactured by Kubota Shoji) at an output of approximately 150 W for approximately 10 minutes at 4°C. The supernatant was then collected by centrifugation. (1-4) The supernatant collected in (1-3) was loaded onto a column packed with IgG Sepharose 6 Fast Flow (GE Healthcare) pre-equilibrated with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl (hereinafter referred to as wash buffer). The column was washed with 10 times the volume of the wash buffer, and then 0.1 M glycine-HCl buffer (pH 3.0) was passed through to collect a fraction corresponding to FcR36i_Cys. The eluted fraction was neutralized with 1 / 50 volume of 1 M Tris-HCl buffer (pH 9.0) to prepare purified FcR36i_Cys. (1-5) The absorbance of the collected fractions was measured using a spectrophotometer to quantify the amount of protein contained in the fractions. SDS-PAGE was also performed to confirm that FcR36i_Cys was highly purified.

[0040] (2) Preparation of FcR36i_Cys immobilized gel (antibody adsorbent) (2-1) Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added to 30 mg of FcR36i_Cys (SEQ ID NO: 4) prepared in (1) to a final concentration of 1 mM. After leaving the mixture to stand for 1 hour, 1.0 g of Haloacetyl Toyopearl (suction-dried gel) prepared in Example 1 and 1 M Tris buffer (pH 9.0) were added and the mixture was allowed to react at 25°C for 2 hours. (2-2) After the reaction was completed, the mixture was washed with 50 mM citrate buffer (pH 3.0), 20 mM MES buffer (pH 6.5), and 20 mM aqueous sodium hydroxide solution in this order. (2-3) To 1.0 g of the washed gel (suction-dried gel), 1.8 mL of 20 mM sodium hydroxide, 57 μmol of α-thioglycerol, and 0.18 mL of 1 M Tris buffer (pH 8.5) were added, and the mixture was allowed to react for an additional 2 hours at 25°C to produce a gel (antibody adsorbent) with immobilized FcR36i_Cys.

[0041] Comparative Example 2 Preparation of antibody adsorbent (part 2) (1) A polypeptide (FcR36i_EAAAK_Cys) (SEQ ID NO: 8) was designed in which a peptide linker consisting of the amino acid sequence set forth in SEQ ID NO: 2 was inserted between the glycine at position 200 and the cysteine ​​at position 201 of FcR36i_Cys (SEQ ID NO: 4).

[0042] (2) PCR was performed using a polynucleotide encoding FcR36i_EAAAK_Cys (SEQ ID NO: 9) as a template. The oligonucleotides used in the PCR were the sequences set forth in SEQ ID NO: 6 and SEQ ID NO: 7. A reaction solution having the composition shown in Table 1 was prepared using the polynucleotide and primers, and the reaction solution was heat-treated at 98°C for 5 minutes. 30 cycles of reaction were performed, each cycle consisting of a first step at 98°C for 10 seconds, a second step at 55°C for 5 seconds, and a third step at 72°C for 1 minute.

[0043] [Table 1]

[0044] (3) The polynucleotide obtained in (2) was purified, digested with the restriction enzymes NcoI and HindIII, and ligated into the expression vector pTrc-PelB (prepared by the method described in WO2015 / 199154) which had been previously digested with the restriction enzymes NcoI and HindIII. The ligation product was used to transform the Escherichia coli W3110 strain.

[0045] (4) The transformant (genetically modified E. coli) obtained in (3) was cultured (37°C, 16 hours) on 2xYT plate medium (peptone 16 g / L, yeast extract 10 g / L, sodium chloride 5 g / L) containing 50 μg / mL of kanamycin, and the expression vector pTrc-FcR36i_EAAAK_Cys was extracted using a QIAprep Spin Miniprep kit (Qiagen).

[0046] (5) The nucleotide sequence of pTrc-FcR36i_EAAAK_Cys was analyzed using an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7. The amino acid sequence of the polypeptide expressed by pTrc-FcR36i_EAAAK_Cys is shown in SEQ ID NO: 8, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 10.

[0047] (6) FcR36i_EAAAK_Cys was prepared from recombinant Escherichia coli containing the expression vector pTrc-FcR36i_EAAAK_Cys obtained in (3) using the method described in Comparative Example 1(1), and a gel (antibody adsorbent) on which FcR36i_EAAAK_Cys was immobilized was prepared using the method described in Comparative Example 1(2).

[0048] Example 2 Preparation of antibody adsorbent (part 3) (1) A polypeptide (FcR36i_(EAAAK)3_Cys) was designed (SEQ ID NO: 11) in which a peptide linker consisting of three repeats of the amino acid sequence set forth in SEQ ID NO: 2 was inserted between the glycine at position 200 and the cysteine ​​at position 201 of FcR36i_Cys (SEQ ID NO: 4).

[0049] (2) PCR was carried out in the same manner as described in Comparative Example 2(2), except that a polynucleotide encoding FcR36i_(EAAAK)3_Cys (SEQ ID NO: 12) was used as the template DNA.

[0050] (3) After purifying the polynucleotide obtained in (2), the polynucleotide was ligated and transformed by the method described in Comparative Example 2(3) to obtain recombinant Escherichia coli, and the expression vector pTrc-FcR36i_(EAAAK)3_Cys was extracted by the method described in Comparative Example 2(4).

[0051] (4) The nucleotide sequence of pTrc-FcR36i_(EAAAK)3_Cys was analyzed by the method described in Comparative Example 2(5). The amino acid sequence of the polypeptide expressed by pTrc-FcR36i_(EAAAK)3_Cys is shown in SEQ ID NO: 11, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 13.

[0052] (5) FcR36i_(EAAAK)3_Cys was prepared from recombinant Escherichia coli containing the expression vector pTrc-FcR36i_(EAAAK)3_Cys obtained in (3) by the method described in Comparative Example 1(1), and a gel (antibody adsorbent) on which FcR36i_(EAAAK)3_Cys was immobilized was prepared by the method described in Comparative Example 1(2).

[0053] Example 3 Preparation of antibody adsorbent (part 4) (1) A polypeptide (FcR36i_(EAAAK)5_Cys) was designed (SEQ ID NO: 14) in which a peptide linker consisting of five repeats of the amino acid sequence set forth in SEQ ID NO: 2 was inserted between the glycine at position 200 and the cysteine ​​at position 201 of FcR36i_Cys (SEQ ID NO: 4).

[0054] (2) PCR was carried out in the same manner as described in Comparative Example 2(2), except that a polynucleotide encoding FcR36i_(EAAAK)5_Cys (SEQ ID NO: 15) was used as the template DNA.

[0055] (3) After purifying the polynucleotide obtained in (2), the polynucleotide was ligated and transformed by the method described in Comparative Example 2(3) to obtain recombinant Escherichia coli, and the expression vector pTrc-FcR36i_(EAAAK)5_Cys was extracted by the method described in Comparative Example 2(4).

[0056] (4) The nucleotide sequence of pTrc-FcR36i_(EAAAK)5_Cys was analyzed by the method described in Comparative Example 2(5). The amino acid sequence of the polypeptide expressed by pTrc-FcR36i_(EAAAK)5_Cys is shown in SEQ ID NO: 14, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 16.

[0057] (5) FcR36i_(EAAAK)5_Cys was prepared from recombinant Escherichia coli containing the expression vector pTrc-FcR36i_(EAAAK)5_Cys obtained in (3) by the method described in Comparative Example 1(1), and a gel (antibody adsorbent) on which FcR36i_(EAAAK)5_Cys was immobilized was prepared by the method described in Comparative Example 1(2).

[0058] Example 4 Preparation of antibody adsorbent (part 5) (1) A polypeptide (FcR36i_D1_Cys) was designed in which a peptide linker consisting of the amino acid sequence set forth in SEQ ID NO: 17, which contains the D1 domain of human FcγRIIIa, was inserted between the 200th glycine and the 201st cysteine ​​of FcR36i_Cys (SEQ ID NO: 4) (SEQ ID NO: 18).

[0059] (2) PCR was performed using a polynucleotide encoding FcR36i_Cys (SEQ ID NO: 5) as template DNA. The primers used in the PCR were either a pair of oligonucleotides consisting of the sequences set forth in SEQ ID NO: 20 and SEQ ID NO: 21 (for vector preparation) or a pair of oligonucleotides consisting of the sequences set forth in SEQ ID NO: 22 and SEQ ID NO: 23 (for insert preparation). A reaction solution having the composition shown in Table 1 was prepared using the template DNA and primers, and the reaction solution was heat-treated at 98°C for 5 minutes. The reaction was then repeated 30 times, with one cycle consisting of a first step at 98°C for 10 seconds, a second step at 55°C for 5 seconds, and a third step at 72°C for 5 minutes.

[0060] (3) The two polynucleotides obtained in (2) were purified, and a mixed solution containing 0.03 pmol of vector polynucleotide and 0.06 pmol of insert polynucleotide was prepared. The vector polynucleotide and the insert polynucleotide were ligated to this mixed solution by Gibson assembly using NEB Builder HiFi DNA Assembly Master Mix (New England BioLabs), and the ligation product was then used to transform Escherichia coli W3110.

[0061] (4) The expression vector pTrc-FcR36i_D1_Cys was extracted from the transformant (genetically modified E. coli) obtained in (3) by the method described in Comparative Example 2(4).

[0062] (5) The nucleotide sequence of pTrc-FcR36i_D1_Cys was analyzed by the method described in Comparative Example 2(5). The amino acid sequence of the polypeptide expressed by pTrc-FcR36i_D1_Cys is shown in SEQ ID NO: 18, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 19.

[0063] (6) FcR36i_D1_Cys was prepared from recombinant Escherichia coli containing the expression vector pTrc-FcR36i_D1_Cys obtained in (3) by the method described in Comparative Example 1(1), and a gel (antibody adsorbent) on which FcR36i_D1_Cys was immobilized was prepared by the method described in Comparative Example 1(2).

[0064] Example 5 Preparation of antibody adsorbent (part 6) (1) A polypeptide (FcR36i_2D1_Cys) was designed (SEQ ID NO: 24) in which a peptide linker consisting of two repeats of the amino acid sequence set forth in SEQ ID NO: 17 was inserted between the 200th glycine and the 201st cysteine ​​of FcR36i_Cys (SEQ ID NO: 4).

[0065] (2) PCR was performed in the same manner as described in Example 4(2), except that a polynucleotide encoding FcR36i_D1_Cys (SEQ ID NO: 19) was used as the template DNA, and either one pair of oligonucleotides consisting of the sequences set forth in SEQ ID NO: 20 and SEQ ID NO: 26 (for vector preparation) or one pair of oligonucleotides consisting of the sequences set forth in SEQ ID NO: 22 and SEQ ID NO: 27 (for insert preparation) was used as PCR primers.

[0066] (3) The two types of polynucleotides obtained in (2) were purified, and then ligated and transformed as described in Example 4(3) to obtain recombinant Escherichia coli, and the expression vector pTrc-FcR36i_2D1_Cys was extracted as described in Comparative Example 2(4).

[0067] (4) The nucleotide sequence of pTrc-FcR36i_2D1_Cys was analyzed by the method described in Comparative Example 2(5). The amino acid sequence of the polypeptide expressed by pTrc-FcR36i_2D1_Cys is shown in SEQ ID NO: 24, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 25.

[0068] (5) FcR36i_2D1_Cys was prepared from recombinant Escherichia coli containing the expression vector pTrc-FcR36i_2D1_Cys obtained in (3) using the method described in Comparative Example 1(1), and a gel (antibody adsorbent) on which FcR36i_2D1_Cys was immobilized was prepared using the method described in Comparative Example 1(2).

[0069] Example 6 Measurement of antibody adsorption amount (part 1) (1) The antibody adsorbents prepared in Comparative Examples 1 and 2 and Examples 2 and 3 were suction-dried by the method described in Example 1 (1-1) to prepare suction-dried gels.

[0070] (2) 150 μL of PBS (Phosphate Buffered Saline) was added to the suction-dried gel, and the gel was centrifuged at 2000 × g for 1 minute. This procedure was repeated three times to wash the gel.

[0071] (3) After washing, 150 μL of PBS and 60 μL of human immunoglobulin solution (globulin intramuscular injection 1500 mg / 10 mL "JB", manufactured by Japan Blood Products Organization) were added sequentially to the gel, and the gel was stirred at 25°C for 2 hours to allow the immunoglobulin (antibody) to be adsorbed onto the gel.

[0072] (4) After the adsorption procedure in (3), the spin column was centrifuged at 2000×g for 1 minute to separate the solution containing unadsorbed antibody from the gel.

[0073] (5) 150 μL of PBS was added to the gel, and the gel was centrifuged at 2000 × g for 1 minute. This procedure was repeated three times to wash the gel.

[0074] (6) 150 μL of 50 mM citrate buffer (pH 3.0) was added to the gel, and the gel was centrifuged at 2000 × g for 1 minute. This procedure was repeated three times to elute the antibody adsorbed to the gel. The absorbance of the eluate was measured to calculate the antibody concentration, and the amount of antibody adsorbed to the Fc receptor-immobilized gel was determined.

[0075] The results are shown in Table 2. The linker lengths in Table 2 are shown for reference based on values ​​described in a literature article (Protein Engineering, 14(8), 529-532 (2001)). It can be seen that the antibody adsorption amount relative to the amount of immobilized FcR for antibody adsorbents with linker lengths of 2 nm or more (Examples 2 and 3) is improved compared to the antibody adsorbent without a linker (Comparative Example 1) and the antibody adsorbent with a linker length of less than 2 nm (Comparative Example 2).

[0076] [Table 2]

[0077] Example 7 Measurement of antibody adsorption amount (part 2) The antibody adsorption amount was measured in the same manner as in Example 6, except that the antibody adsorbents prepared in Comparative Example 1 and Examples 4 and 5 were used instead of the antibody adsorbent in Example 6(1).

[0078] The results are shown in Table 3. The linker length in Table 3 refers to the approximate length of the D1 domain of FcγRIIIa, calculated from the crystal structure (Protein Databank: 3AY4) containing the Fc receptor. Even if the peptide linker sequence is not a simple repetition of an amino acid sequence as in Examples 2 and 3, but is a domain sequence of a specific protein, it can be seen that as long as the linker length is 2 nm or longer, the amount of antibody adsorption relative to the amount of FcR immobilized is improved compared to an antibody adsorbent without a linker (Comparative Example 1).

[0079] [Table 3]

Claims

1. An antibody adsorbent comprising an Fc receptor, an insoluble carrier, and a peptide linker for immobilizing the receptor on the carrier, The Fc receptor is a polypeptide comprising at least the amino acid residues from glycine at position 24 to glutamine at position 199 of the amino acid sequence set forth in SEQ ID NO:4, The peptide linker is a polypeptide represented by any one of the following (1) or (2): (1) A polypeptide comprising at least the amino acid residues from the 6th proline to the 87th histidine in the amino acid sequence set forth in SEQ ID NO: 17; (2) A polypeptide consisting of a sequence in which the amino acid sequence set forth in SEQ ID NO:2 is repeated three or more times. and The adsorbent, wherein the length of the peptide linker is 2 nm or more.

2. 13. A method for purifying an antibody, comprising the steps of: adding a solution containing an antibody to a column packed with the adsorbent according to claim 1 to adsorb the antibody onto the adsorbent; and eluting the antibody adsorbed onto the adsorbent using an elution solution.

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